S123: DECODING TRANSCRIPTOMIC AND EPIGENETIC CONSEQUENCES OF STRUCTURAL VARIANTS IN CK-AML AT SINGLE-CELL RESOLUTION
Authors: A.-M. Leppä, K. Grimes, H. Jeong, T. Boch, D Karpova, A. Jauch, F. Grünschläger, A. Dolnik, L. Bullinger, A. Krämer, A. D. Sanders, J. O. Korbel, A. Trumpp
Journal: HemaSphere (2022)

Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is a heterogenous AML subgroup with dismal response to standard treatment. CK-AML has remained poorly characterized at the molecular level, with mainstream techniques struggling to unravel the complexity of the structural variants (SVs) and link them to phenotypic characteristics. To improve our understanding of molecular dynamics in CK-AML, we established an integrated single cell multi-omics approach that combines the analysis of SVs and nucleosome occupancy (NO) profiling (scNOVA) with concurrent immunophenotypic and transcriptomic profiling (CITE-seq). Aims: To characterize the genetic and non-genetic intra-patient heterogeneity at single cell resolution in CK-AML. Methods: For scNOVA analysis, blasts from four CK-AML patients were cultured in the presence of BrdU for the duration of one cell division, followed by single-cell template strand-sequencing (Strand-seq). For CITE-seq analysis, cells were stained with 38 antibody-derived tags for combined analysis of single cell transcriptome and immunophenotype. For functional studies, xenografts of the same CK-AML patients were generated by injection of AML cells into the femoral BM cavity of sublethally irradiated NOD.Prkdcscid.Il2rgnull (NSG) mice. Results: To characterize cellular hierarchies together with epigenetic, transcriptomic, and surface protein features of CK-AML, we applied an integrated single-cell analytical framework, termed scNOVA-CITE, on over 15,000 leukemic cells from four diagnostic CK-AML patient samples. We detected complex clonal evolution patterns driven by simple and complex SVs in all patients. Three out of four patients harbored SVs leading to loss of one TP53 wild-type allele, often accompanied by loss-of-function mutations in the remaining allele. One patient showed parallel evolution of TP53-deleted and TP53 wild-type subclones in the same sample, with the latter clone harboring a lower abundance of SVs compared to the TP53-deleted cells. Moreover, based on NO we inferred lowest activity of TP53 pathway in the cells with TP53 aberrations, emphasizing the role of TP53 in intra-patient cytogenetic evolution as well as inter-patient heterogeneity. Thereby, we unraveled novel single-cell SV landscapes in CK-AML and explored their epigenetic impact. To assess in depth the functional outcomes of the genetic complexity, we also characterized the transcriptome and cell surface proteome using scNOVA-CITE. Two patients showed high genetic intra-patient heterogeneity and harbored multiple subclones at diagnosis with unique transcriptomic features. When transplanted into NSG mice, we detected predominantly monoclonal engraftment and strongly reduced genomic instability in the respective patient-derived xenografts. The engrafting clones could be traced back to a small subclone in the original patient samples. Molecular analysis of the subclones in the diagnostic samples by scNOVA-CITE revealed converging characteristics leading to stem-like phenotypes. Taken together, these data indicate that cytogenetic evolution resulting in stemness phenotype in CK-AML is advantageous for leukemic stem cell expansion in mice, unfolding novel ways to study regulation of stemness and to identify potential therapeutic targets. Summary/Conclusion: By developing scNOVA-CITE, we established an integrative single-cell multi-omics framework allowing to characterize genetic, epigenetic, transcriptomic and cell surface proteomic properties of CK-AML patient cells. We provide unique insights into the genetic complexity of CK-AML, and evidence of the resulting functional outcomes.